Paul K. Herman - US grants

DESCRIPTION (provided by applicant): When conditions are not optimal for continued growth, cells stop dividing and enter into a specialized resting state, known as G0 in mammals and stationary phase in bacteria and yeasts. Cells in these quiescent states exhibit an increased resistance to environmental stress and can remain viable, although not dividing, for very long periods of time. Moreover, the transitions between these periods of quiescence and the mitotic cycle have been shown to be key points of proliferative control. Therefore, an understanding of the biology of the resting cell is an essential requirement for a complete description of the mechanisms governing eukaryotic cell proliferation. This proposal is focused on the mechanisms regulating the entry into stationary phase in the budding yeast, Saccharomyces cerevisiae. In particular, the proposed experiments focus on a collection of mutants that are defective for stationary phase entry. Several of the genes identified by these rye mutants have been found to encode important regulators of RNA polymerase (pol) II activity. Moreover, studies of the rye mutants have suggested that RNA pol II activity is controlled by the Ras protein signaling pathway. This signal transduction pathway is a key regulator of cell growth in most, if not all, eukaryotes. Interestingly, these Ras effects appear to be mediated by the proteins encoded by the above RYE genes. Therefore, the Rye proteins could be an essential link between Ras signaling and RNA pol II that allows for the proper coordination of growth and gene expression. The above work has suggested a novel mode of transcriptional control whereby signaling pathways regulate gene expression by directly targeting proteins within the RNA pol II holoenzyme. This proposal will test this hypothesis by examining whether particular Rye proteins are indeed targeted by the Ras signaling pathway. In addition, the proposed experiments will examine the roles of the Rye proteins in stationary phase entry and how these roles are affected by Ras signaling activity. Finally, the remaining rye mutants will be characterized as well as the gene expression changes that occur upon the entry into stationary phase. In all, the completion of the proposed experiments should provide insights into the role of Ras signaling activity during stationary phase entry and the manner in which growth control is coordinated with the regulation of gene expression.

DESCRIPTION (provided by applicant): We are interested in developing a better understanding of the biology of the non-dividing resting states that eukaryotic cells enter when conditions are not conducive to continued growth. One of our primary goals is to define how the processes that are induced during these periods of quiescence are regulated and how they contribute to general cell survival. This proposal is focused upon a set of related pathways, known collectively as autophagy, that are required for this survival. Autophagy pathways are responsible for the turnover of cytoplasmic material, including bulk protein and damaged or superfluous organelles. This autophagy-mediated degradation has been linked to a variety of processes relevant to human health, including tumor suppression, innate immunity and neurological disorders, such as Huntington's disease. In many of these conditions, the autophagy pathway is being considered as a major point of therapeutic intervention. It is therefore critical that we develop a thorough understanding of the mechanisms normally controlling autophagy in eukaryotic cells. This proposal will examine two key aspects of the regulation of autophagy in the yeast, Saccharomyces cerevisiae. Studies with this organism have contributed tremendously to our basic understanding of autophagy in all eukaryotes, including humans. First, a combination of approaches will be used to characterize the autophagy process induced by the inactivation of the cAMP-dependent protein kinase (PKA) signaling pathway. Our preliminary work indicates that this PKA-regulated process may be similar to an alternative form of macroautophagy recently identified in mammals. The experiments here will explore this possibility and define the molecular machineries governing this PKA-regulated pathway. These studies will also characterize a potential role for the phosphoinositide, PtdIns (3,5)P2, in this PKA-regulated macroautophagy. The second major goal of this proposal is the identification of the substrates of the Atg1 protein kinase. Atg1 is a key regulatory target within the autophagy machinery and identifying the targets of this enzyme represents one of the major goals in the autophagy field today. In all, we feel that the completion of this work will provide important insights into the manner in which autophagy is controlled in eukaryotic cells, insights that should facilitate efforts to manipulate this pathway in clinically beneficial ways. The specific aims in this proposal are: (1) to characterize the autophagy pathway induced upon inactivation of PKA signaling;(2) to characterize the link between PKA signaling, phosphoinositide metabolism and the regulation of autophagy;and (3) to identify and characterize substrates of the Atg1 protein kinase. PUBLIC HEALTH RELEVANCE: This proposal aims to further our understanding of a process known as autophagy that has been linked to a number of serious human ailments, including breast and ovarian cancer, Crohn's disease and neurological disorders, such as Huntington's disease. Interestingly, drugs that target autophagy are being developed as potential therapeutics for many of these conditions. By increasing our understanding of the normal control of the autophagy process, the work here would provide potentially novel avenues for this drug discovery process.

DESCRIPTION (provided by applicant): We are interested in the biology of the G0-like resting states that eukaryotic cells enter when conditions are not conducive to continued growth. Our goal is to define the processes that are induced during these periods of quiescence and to determine how they collectively contribute to cell survival. This proposal extends this analysis and examines a particular ribonucleoprotein (RNP) complex that forms in the cytoplasm of resting cells, known as a Processing-body, or P-body. P-bodies are relatively large aggregate structures that contain non-translating mRNAs and a distinct set of protein constituents, including a number of enzymes involved in the processing of these transcripts. Interestingly, the P-body is just one of a large number of similar cytoplasmic structures that form in the G0 cell. This prevalence suggests that these complexes are important for the biology of the resting cell, but little is presently known about the underlying reasons for this large-scale sequestration of macromolecules. These cytoplasmic structures also appear to be important for human health as a related RNP complex, the stress granule, has been implicated in the pathology of neurological disorders, like amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2. In all, thes data suggest that these cytoplasmic foci play important and diverse roles in eukaryotic biology. It is therefore critical that we develop a better understanding of the functions of these structure and the manner in which their assembly is regulated. This proposal addresses these broader issues by examining a model RNP complex, the P-body of the yeast, Saccharomyces cerevisiae. P-bodies have been conserved from yeast to humans, and much of what we know about these RNP structures has come from studies with this budding yeast. However, despite extensive effort, little was known about the mechanisms regulating P-body assembly and the physiological role of the larger foci that form in quiescent cells. Our recent work has suggested interesting answers to both of these questions. In particular, we have identified the cAMP-dependent protein kinase (PKA) as a key regulator of P- body assembly, and found that the larger P-body foci appear to be required for the long-term survival of quiescent cells. Our data indicate that PKA directly phosphorylates Pat1, a conserved core constituent of P- bodies, and thereby disrupts the formation of the larger aggregate structures. The experiments proposed here aim to define the mechanistic details of this control by PKA and to elucidate how the larger foci promote cell viability. Finally, we will explore further the intriguing observation that signaing molecules, like PKA itself, are also found in P-bodies and related complexes. The underlying hypothesis to be tested is that specific proteins and mRNAs required for the subsequent resumption of growth are stored and protected within P-body foci in quiescent cells. The specific aims of this proposal are: (1) to determine the role of PKA signaling in the regulation of P-body foci formation; and (2) to determine the physiological role of P-body foci in quiescent cells.

The eukaryotic cell is a highly compartmentalized structure that is subdivided into discrete functional areas by the presence of a variety of membrane-enclosed organelles. This segregation of functions is essential for normal cell growth and survival. Interestingly, recent studies have indicated that additional levels of compartmentalization exist within these cells. In particular, a number of cytoplasmic granules that contain distinct sets of proteins and mRNAs have been identified. Two of the best-characterized of these ribonucleoprotein (RNP) structures are the Processing-body (P-body) and stress granule. These granules differ from the more traditional organelles in that they lack a limiting membrane and are rather dynamic in nature. These granules are evolutionarily conserved and have been linked to a number of human diseases, including a variety of cancers and neurodegenerative disorders. However, despite these observations, the physiological functions of these RNP structures remain poorly understood. This lack of understanding represents a critical gap in our current knowledge and attempting to bridge this divide is a primary research focus in our lab. The experiments in this proposal aim to further our understanding of both the biological roles of these RNP granules and the mechanisms that regulate their assembly. The first two aims come at this question of biological function from different directions. In the first, we will assess the physiological consequences of having key signaling proteins associate with the P-body and/or stress granule during conditions of stress. Our focus here is on particular protein kinases and the possibility that this re-localization to RNP granules allows for a rewiring of the signaling networks present in the cell. In the second, we focus on the granule as a whole and ask how cell physiology is altered in mutants that lack these RNP structures. These latter studies focus on a potential role in protein homeostasis that was suggested by recent work from our lab. In particular, we have found that mutants lacking P-bodies exhibit elevated levels of protein misfolding and aggregation. The experiments here aim to determine the underlying mechanisms responsible for these effects and should establish whether these RNP granules have a direct role in the maintenance of protein quality control (PQC). Finally, the third aim examines several key aspects of the regulation and assembly of P-body foci. In particular, the studies will define the molecular mechanism underlying PKA-mediated control of P-body assembly and develop a facile method for the purification of these RNP granules. The three specific aims of the proposal are as follows: (1) determine the physiological consequences of protein kinase recruitment to RNP granules; (2) define the underlying mechanisms responsible for the P-body-mediated effects on PQC; and (3) examine the process and regulation of P-body assembly.